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2308 Part XII: Hemostasis and Thrombosis Chapter 135: Fibrinolysis and Thrombolysis 2309

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of platelet α granules. Plasmin released into flowing blood or in the both a Mr 47,000 nonglycosylated intracellular form and an Mr 60,000
vicinity of a platelet–rich thrombus, is immediately neutralized upon glycosylated form secreted by leukocytes and fibrosarcoma cells. Func-
forming an irreversible 1:1 stoichiometric, lysine-binding site–dependent tionally, PAI–2 inhibits both two–chain t-PA and two–chain u-PA with
5
–1 –1
complex with α -PI. Interaction with plasmin is accompanied by comparable efficiency (second order rate constants 10 M s ). How-
2
cleavage of the Arg364–Met365 peptide bond, and the resulting cova- ever, it is less effective toward single-chain t-PA (second order rate con-
–1 –1
3
lent complexes are cleared in the liver. Mice globally deficient in α -PI stant 10 M s ), and does not inhibit prourokinase.
2
display reduced fibrin deposition, following treatment with endotoxin Significant levels of PAI–2 are found in human plasma primarily
and enhanced lysis of injected plasma clots, but no spontaneous bleeding during pregnancy. The gene’s 5′–untranslated region contains a potent
(see Table 35–2). 90 silencer, the PAUSE-1 element, which may be responsible for its low
Several additional proteins can act as plasmin inhibitors (see level of expression in nonpregnant individuals. 127,128 The 3′–downstream
Table 135–1). α –Macroglobulin is a Mr 725,000 dimeric protein synthe- sequences include the TTATTTAT motif which has been identified
2
sized by endothelial cells and macrophages, and found in platelet α gran- with inflammatory mediators. 129,130 In macrophages in vitro, secretion
ules. This nonserpin inhibits plasmin with approximately 10 percent of of PAI-2 is enhanced by endotoxin and phorbol esters 130,131 and dex-
the efficiency exhibited by α –PI by forming noncovalent complexes with amethasone decreases PAI–2 expression in HT–1080 cells. 55
2
91
several distinct serine proteases. C -esterase inhibitor can inhibit t-PA in
1
plasma and protease nexin may function as a noncirculating cell surface Thrombin-Activatable Fibrinolysis Inhibitor
92
inhibitor of trypsin, thrombin, factor Xa, urokinase, or plasmin, result- Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxy-
ing in protease–inhibitor complexes that are endocytosed via a specific peptidase with specificity for carboxy terminal arginine and lysine resi-
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nexin receptor. 93,94 The purpose of these multiple plasmin inhibitors is to dues. The action of TAFI eliminates binding sites for Plg and t-PA on
guard against premature plasmin activation and subsequent degradation fibrin. This single-chain Mr 60,000 polypeptide circulates in plasma at
133
of fibrinogen, until intravascular fibrin begins to appear/. concentrations of approximately 75 nM, and undergoes limited prote-
olysis in the presence of thrombin, which leads to its activation. 134–136
Plasminogen Activator Inhibitors The profibrinolytic effect of activated protein C in plasma is a result of
Plasminogen Activator Inhibitor-1 Of the two major Plg activa- its ability to inactivate coagulation factors Va and VIIIa, which reduces
95
tor inhibitors, PAI–1 is the most ubiquitous (see Table 135–1). This activation of thrombin, the primary activator of TAFI. The profi-
132
Mr approximately 52,000 single–chain, cysteine–less glycoprotein is brinolytic effect of activated protein C in an in vitro plasma-based sys-
released by endothelial cells, monocytes, macrophages, hepatocytes, tem was TAFI-dependent, and, in a system of purified components,
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adipocytes, and platelets. 96–98 Release of PAI-1 is stimulated by many TAFI has been shown to downregulate t-PA–induced fibrinolysis half-
cytokines, growth factors, and lipoproteins common to the global maximally at a concentration of approximately 1 nM, which is 2 percent
inflammatory response. 69,70,99,100,101 The PAI-1 gene consists of nine of its concentration in plasma. Inhibition of either the intrinsic path-
138
102
exons, spanning 12.2 kb on chromosome 7q21.3–q22. The serpin- way of coagulation or TAFI itself results in a doubling of endogenous clot
reactive site is located at Arg346–Met347, and activity of this labile lysis in an in vivo rabbit jugular vein model of thrombolysis. 139,140 TAFI-
serpin is stabilized upon complex formation with vitronectin, a compo- deficient mice display increased lysis of plasma clots and reduced injury-
nent of plasma and pericellular matrix. 103–105 induced venous thrombosis (see Table 135–2). 141,142 In plasma, TAFI may
Regulation of PAI-1 gene expression is complex. 106,107 The upstream regulate Plg binding to both cell surface receptors and to fibrin. 143
regulatory region of the human PAI-1 gene contains a strong endothe-
lial cell/fibroblast-specific element 108,109 a glucocorticoid–responsive
109
110
enhancer, and TGF-β responsive elements. TGF-β is known to CELLULAR RECEPTORS
stimulate fos and jun, the two components of the AP1 complex, and A large number of structurally diverse fibrinolytic “activation” and “clear-
an AP1 binding site (GGAGTCA) is located upstream of the PAI–1 cap ance” receptors have been described. Here, we focus on endothelial cell
site. Agents shown to enhance expression of PAI–1 at the message activation receptors that are likely to contribute to homeostatic control
111
2
level, the protein level, or both, without affecting t-PA synthesis, include of plasmin activity (see Table 135–1). Clearance receptors eliminate
the inflammatory cytokines lipopolysaccharide, IL–1, tumor necrosis plasmin and Plg activators from the blood or focal microenvironments.
factor–α, 69,70,99,112,113 TGF-β and basic fibroblast growth factor, 71,99,110,114
very-low-density lipoprotein and lipoprotein(a), 115,116 angiotensin II, Activation Receptors
117
120
thrombin, 118,119 and phorbol esters. In addition, endothelial cell PAI–1 Plasminogen Receptors Proposed Plg receptors include α–enolase,
is downregulated by forskolin and by endothelial cell growth factor in glycoprotein IIb/IIIa complex, the Heymann nephritis antigen,
56
the presence of heparin. 121 amphoterin, the annexin A2/S100A10 complex, histone H2B, and
PAI-1 is the most important and rapidly acting physiologic inhib- plasminogen receptor-KT (Plg-R ) ; these are expressed on a wide
2,3
KT
itor of both t-PA and u-PA. Transgenic mice that overexpress PAI–1 spectrum of cells, including monocytoid cells, platelets, renal epithe-
exhibit thrombotic occlusion of tail veins and swelling of hind limbs lial cells, neuroblastoma cells, endothelial cells, and tumor cells. 144–151
within 2 weeks of birth. Mice deficient in PAI–1, on the other hand, Typically, Plg receptors interact with the kringle structures of Plg through
122
exhibit normal fertility, viability, tissue histology, and development, and carboxyl–terminal lysine residues that are either present on the native
are resistant to endotoxin-induced thrombosis, but show no evidence protein, or generated by limited proteolysis. 144
of overt hemorrhage (see Table 135–2). 123,124 These observations con- Urokinase Plasminogen Activator Receptor The u-PA receptor
trast with the moderately severe bleeding disorder observed in a human (uPAR) is expressed on monocytes, macrophages, fibroblasts, endo-
patient with complete PAI-1 deficiency. 125 thelial cells, and many tumor cells (see Table 135–1). 152,153 uPAR com-
Plasminogen Activator Inhibitor-2 Originally purified from plementary DNA (cDNA) was cloned and sequenced from a human
human placenta, PAI-2 is a 393-amino-acid member of the serpin fibroblast cDNA library and encodes a protein of 313 amino acids
154
126
family whose reactive site is the Arg358–Thr359 peptide bond (see with a 21–residue signal peptide. The gene consists of seven exons dis-
Table 135–1). The gene encoding PAI–2 is located on chromosome tributed over 23 kb of genomic DNA, and places this glycoprotein within
127
18q21–23, spans 16.5 kb, and contains eight exons. PAI–2 exists as the Ly-1/elapid venom toxin superfamily of cysteine rich proteins. 155,156
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